The Present Invention Relates To Enzyme Immobilization On Fibrous Matrices And, More Particularly, To Immobilization Of B-Galactosidase On A Fibrous Matrix For Production Of Galacto-Oligosaccharides From Lactose.
Biocatalyst immobilization is gaining increased attention for the synthesis of industrial bioproducts ranging from neutraceuticals to chemicals. Enzyme immobilization provides many important advantages over use of enzymes in soluble form, namely, enzyme reusability, continuous operation, controlled product formation, and simplified and efficient processing. The main challenges in enzyme immobilization include not only containment of a large amount of enzyme to be immobilized while retaining most of its initial activity, but also the performance of immobilized enzyme in actual production processes in industrial-type reactors. Thus, the success of immobilized enzyme is not only driven by its applications but also relies on a number of factors, including enzyme support, chemical reagent, and reactor.
Enzyme support is generally considered as the most important component contributing to the performance of the immobilized biocatalyst reactor. In addition to being a very inexpensive and widely available fibrous material, cotton cloth provides a number of desirable characteristics, including high porosity (>95%), large specific surface area, and excellent mechanical strength. Cotton cloth has been successfully used in cell immobilization and fermentation studies. Cotton cloth immobilized enzyme placed in a loose spiral shape in a plug-flow-type reactor provides good flow rates, low pressure drop, and negligible mass transfer resistance. These characteristics are also highly desirable for industrial enzyme application. Thus, cotton fabric also can be used for the development of an industrially applicable fibrous bed enzyme bioreactor where the immobilized enzyme functions as good as soluble enzyme.
Although enzymes can be immobilized on cotton cloth activated with tosyl chloride, the method is somewhat tedious and involves the use of organic chemicals. Polyethyleneimine (PEI), an extremely branched cationic chain polymer, has many applications in biochemistry because of its electrostatic interaction with negatively charged species. PEI has been an essential ingredient of many enzyme immobilization procedures, where it serves to coat an inert support such as porous glass microbeads or charged insoluble carriers. Cotton cloth coated with PEI has been used as a support for immobilization of several enzymes, including glucose oxidase, urease, and invertase, and yeast cells. In these applications, PEI is adsorbed on the cotton cloth and then excess PEI is washed away with water or buffer solution. The remaining PEI is then cross-linked with glutaraldehyde before and/or after enzyme coupling. However, the amount of enzyme immobilized is rather low and needs to be improved for industrial applications.
Lactose found in cheese whey is an abundant byproduct from the dairy industry and can be used to produce galacto-oligosaccharides (GOS), a prebiotic functional food ingredient that selectively stimulates the growth of bifidobacteria in the lower part of the human intestine. Commercial potential for applications of galacto-oligosaccharides in food product lines is high because of its many health benefits, but an economical production process still needs to be developed. There has been a steady 3% annual increase in cheese production. The already problematic lactose is thus expected to be a major concern for the dairy industry. Although there has been extensive research for better utilization of whey lactose, the dairy industry is still in need of new technologies for converting lactose into marketable products. Thus, converting lactose into a valuable food ingredient such as galacto-oligosaccharides that is free of problems associated with lactose is of benefit and highly desirable by the food industry.
Production of galacto-oligosaccharides by immobilized β-galactosidase has been considered in several studies. However, galacto-oligosaccharide production from immobilized enzymes has not been addressed very well. Many of the carriers used for immobilization of β-galactosidases applied in galacto-oligosaccharide production are types of microparticles, such as ion exchange resins, chitosan beads, cellulose beads, and agarose beads. In addition to operational (back pressure, aggregation, clogging) and economical (expensive) disadvantages, commonly noted diffusion limitations in these immobilized systems not only reduce the reaction rate in general but also affect the product spectrum and specifically reduce galacto-oligosaccharide formation. For example, 20-30% decreases in the galacto-oligosaccharide formation have been reported with immobilized enzymes due to introduction of mass transfer resistance in the system.
Accordingly, there is a recognized need for improvements in methods of enzyme immobilization on fibrous matrices design.